While there have been advances in cancer treatment, chemotherapy remains largely inefficient and ineffective. One reason for the generally poor performance of chemotherapy is that the selected treatment is often not closely matched to the genetic and molecular dependencies of an individual's disease.
For example, cancer cells exhibit abnormalities, such as DNA damage, genetic instability, abnormal growth factor signaling, and abnormal or missing matrix interactions, any of which should typically induce apoptosis through the intrinsic (mitochondrial) apoptosis pathway. As a result of these aberrant phenotypes, cancer cells develop blocks in apoptosis pathways that allow the cells to survive rather than respond to the apoptosis signals. As many cancer therapies rely on apoptosis to be effective, modulation of apoptosis by a specific anti-apoptotic protein may relate to responsiveness to particular therapy.
The concept of “oncogene addiction” describes the phenomena of the acquired dependence of cancer cells on, or addiction to, particular proteins for survival. These dependencies make some cancer cells both resistant to particular therapies, and, surprisingly, sensitive to other therapies.
Dependence by cancer cells on the anti-apoptotic Bcl-2 family proteins frequently relates to their otherwise unintended survival. Cancer cells generally rely on one Bcl-2 family member or another (e.g. Bcl-2, Bcl-xL, Mcl-1) to suppress cell death signals and resist apoptosis. Bcl-2 family proteins are regulated by distinct protein-protein interactions between pro-survival (i.e., anti-apoptotic) and pro-apoptotic members. These interactions occur primarily through Bcl-2 homology domain-3 (BH3) mediated binding and can have various outcomes, including homeostasis, cell death, sensitization to apoptosis, and blockade of apoptosis. Many cancer cells in which apoptotic signaling is blocked have an accumulation of the BH3 only activator proteins at the mitochondrial surface, a result of these proteins being sequestered by the anti-apoptotic proteins. This accumulation and proximity to their effector target proteins accounts for increased sensitivity to antagonism of Bcl-2 family proteins in the “primed” state. Accordingly, measurement of the functionality of anti-apoptotic Bcl-2 family proteins have proven to provide sound predictions for the dependency cancer cells have on a given Bcl-2 family member and how a cancer subject will respond to a treatment.
There are two main profiling assays currently used for BH3 profiling. The primary difference in the two commonly used assays is the use of flow cytometry to measure the response versus a fluorescence microplate reader. Using flow cytometry requires fluorescently labeling the cells with antibodies directed toward various cell surface markers and using the gating functions on the flow cytometer to only measure the response in the malignant population of cells. In short, after isolating leukocytes from a sample, the cells are labeled, the outer membrane is permeabilized, contacted with a BH3 peptide (e.g., NOXA), and stained with JC-1 fluorescent dye. Then, flow cytometry is used to quantify the response.
Alternatively, a microplate reader uses cell surface marker antibodies and cell separation techniques to isolate the malignant population of cells. After isolating leukocytes from a sample, the cells of interest are purified, the outer membrane is permeabilized, contacted with a BH3 peptide (e.g., NOXA), and stained with JC-1 fluorescent dye. Then, a microplate reader is used to quantify the response.
Both approaches generally require the cancer cells to be permeabilized by digitonin, which allows fragments of Bcl-2 family peptides (such as NOXA, BIM, etc.) to enter the cell and interact with mitochondrial proteins. In most assays using the standard NOXA peptide, this step is essential. However, cell permeabilization adds complexity, introduces significant variation to the assay, and increases the overall assay run time, all of which introduce technical challenges to providing accurate profiling results that are cost effective. As digitonin non-selectively permeabilizes biological membranes, including the mitochondrial membrane (Hoppel, C, and Cooper, C. Biochem J. 1968 April; 107(3): 367375), the assays that use digitonin generally require precise titration of the digitonin such that the concentration used is within the window that permeabilizes the outer cellular membrane with minimal effects on the mitochondrial membrane. This window of digitonin concentration and treatment time is narrow, may vary between different cell types, and is directly related to having a robust assay that produces accurate results. This challenge is traditionally overcome currently by performing the assay at a single central laboratory that has the experience and the appropriate controls to ensure the assay is performed correctly. Thus, the cell permeabilization step is a challenge for decentralizing the use of such assays, for example, in producing an in vitro diagnostic kit that may be used in clinical laboratories.
Accordingly, improved methods of measuring the functionality of anti-apoptotic Bcl-2 proteins that are more accurate, reproducible, and cost-effective are needed.